Multiply-substituted protease variants with altered net charge for use in detergents

ABSTRACT

Novel protease variants derived from the DNA sequences of naturally-occurring or recombinant non-human proteases are disclosed. The variant proteases, in general, are obtained by in vitro modification of a precursor DNA sequence encoding the naturally-occurring or recombinant protease to generate the substitution of a plurality of amino acid residues in the amino acid sequence of a precursor protease. Protease variants are provided that contain substitutions of the amino acids at one or more residue positions so that the substitution alters the charge at that position to make the charge more negative or less positive compared to a precursor protease and thus the protease variant is more effective in a low detergent concentration system than a precursor protease. Also provided are protease variants containing substitutions of the amino acids at one or more residue positions so that the substitution alters the charge at that position to make the charge more positive or less negative compared to a precursor protease and thus the protease variant is more effective in a high detergent concentration system than a precursor protease. 
     Protease variants are provided that contain substitutions of the amino acids at one or more residue positions so that the substitution alters the charge at that position to make the charge more negative or less positive compared to a precursor protease and thus the protease variant is more effective in a medium detergent concentration system than a precursor protease. Also provided are protease variants containing substitutions of the amino acids at one or more residue positions so that the substitution alters the charge at that position to make the charge more positive or less negative compared to a precursor protease and thus the protease variant is more effective in a medium detergent concentration system than a precursor protease. 
     Further provided is a method of producing a protease variant that is more effective in a low detergent concentration system, medium detergent concentration system and high detergent concentration system than a precursor protease.

RELATED APPLICATIONS

The present application is a Continuation of U.S. patent applicationSer. No. 09/177,353, filed Oct. 23, 1998, now U.S. Pat. No. 6,673,590,issued Jan. 6, 2004, which is a Continuation-in-Part of U.S. patentapplication Ser. No. 08/956,323, filed Oct. 23, 1997, now abandoned, andis a Continuation-in-Part of U.S. patent application Ser. No.08/956,564, filed Oct. 23, 1997, now abandoned, and aContinuation-in-Part of U.S. patent application Ser. No. 08/956,324,filed Oct. 23, 1997, now abandoned.

BACKGROUND OF THE INVENTION

Serine proteases they comprise a diverse class of enzymes having a widerange of specificities and biological functions. Stroud, R. Sci. Amer.,131:74–85. Despite their functional diversity, the catalytic machineryof serine proteases has been approached by at least two geneticallydistinct families of enzymes: 1) the subtilisins and 2) the mammalianchymotrypsin-related and homologous bacterial serine proteases (e.g.,trypsin and S. gresius trypsin). These two families of serine proteasesshow remarkably similar mechanisms of catalysis. Kraut. J. (1977), Annu.Rev. Biochem, 46:331–358. Furthermore, although the primary structure isunrelated, the tertiary structure of these two enzyme families bringstogether a conserved catalytic triad of amino acids consisting ofserine, histidine and aspartate.

Subtilisins are serine proteases (approx. MW 27,500) which are secretedin large amounts from a wide variety of Bacillus species and othermicroorganisms. The protein sequence of subtilisin has been determinedfrom at least nine different species of Bacillus. Markland, F. S., etal. (1983), Hoppe-Seyler's Z. Physiol. Chem., 364:1537–1540. Thethree-dimensional crystallographic structure of subtilisins fromBacillus amyloliquefaciens, Bacillus licheniformis and several naturalvariants of B. lentus have been reported. These studies indicate thatalthough subtilisin is genetically unrelated to the mammalian serineproteases, it has a similar active site structure. The x-ray crystalstructures of subtilisin containing covalently bound peptide inhibitors(Robertus, J. D. et al. (1972), Biochemistry, 11:2439–2449) or productcomplexes (POULOS, et al. (1976), J. Biol. Chem., 251:1097–1103) havealso provided information regarding the active site and putativesubstrate binding cleft of subtilisin. In addition, a large number ofkinetic and chemical modification studies have been reported forsubtilisin; Svendsen, B. (1976), Carlsberg Res. Commun., 41:237–291;Markland, F. S. Id.) as well as at least one report wherein the sidechain of methionine at residue 222 of subtilisin was converted byhydrogen peroxide to methionine-sulfoxide (Stauffer, D. C., et al.(1965), J. Biol. Chem., 244:5333–5338) and extensive site-specificmutagenesis has been carried out (Wells and Estell (1988) TIBS13:291–297)

A common issue in the development of a protease variant for use in adetergent formulation is the variety of wash conditions includingvarying detergent formulations that a protease variant might be used in.For example, detergent formulations used in different areas havedifferent concentrations of their relevant components present in thewash water. For example, a European detergent system typically has about4500–5000 ppm of detergent components in the wash water while a Japanesedetergent system typically has approximately 667 ppm of detergentcomponents in the wash water. In North America, particularly the UnitedStates, a detergent system typically has about 975 ppm of detergentcomponents present in the wash water. Surprisingly, a method for therational design of a protease variant for use in a low detergentconcentration system, a high detergent concentration system, and/or amedium detergent concentration system as well as for use in all threetypes of detergent concentration systems has been developed.

SUMMARY OF THE INVENTION

It is an object herein to provide protease variants containingsubstitutions of the amino acids at one or more residue positions sothat the substitution alters the charge at that position to make thecharge more negative or less positive compared to a precursor proteaseand thus the protease variant is more effective in a low detergentconcentration system than a precursor protease. A low detergentconcentration system is a wash system that has less than about 800 ppmof detergent components present in the wash water.

It is another object herein to provide protease variants containingsubstitutions of the amino acids at one or more residue positions sothat the substitution alters the charge at that position to make thecharge more positive or less negative compared to a precursor proteaseand thus the protease variant is more effective in a high detergentconcentration system than a precursor protease. A high detergentconcentration system is a wash system that has greater than about 2000ppm of detergent components present in the wash water.

It is another object herein to provide protease variants containingsubstitutions of the amino acids at one or more residue positions sothat the substitution alters the charge at that position to make thecharge more positive or less negative compared to a precursor proteaseand thus the protease variant is more effective in a medium detergentconcentration system than a precursor protease. A medium detergentconcentration system is a system that has between about 800 ppm andabout 2000 ppm of detergent components present in the wash water.

It is another object herein to provide protease variants containingsubstitutions of the amino acids at one or more residue positions sothat the substitution alters the charge at that position to make thecharge more negative or less positive compared to a precursor proteaseand thus the protease variant is more effective in a medium detergentconcentration system than a precursor protease. A medium detergentconcentration system is a wash system that has between about 800 ppm toabout 2000 ppm of detergent components present in the wash water.

It is a further object to provide DNA sequences encoding such proteasevariants, as well as expression vectors containing such variant DNAsequences.

Still further, another object of the invention is to provide host cellstransformed with such vectors, as well as host cells which are capableof expressing such DNA to produce protease variants eitherintracellularly or extracellularly.

There is further provided a cleaning composition comprising a proteasevariant of the present invention.

Additionally, there is provided an animal feed comprising a proteasevariant of the present invention.

Also provided is a composition for the treatment of a textile comprisinga protease variant of the present invention.

There is further provided a method of producing a protease variant thatis more effective in a low, medium and high detergent concentrationsystem than a precursor protease including:

-   -   a) substituting an amino acid at one or more residue positions        wherein the substitution alters the charge at that position to        make the charge more positive or less negative compared to the        precursor protease;    -   b) substituting an amino acid at one or more residue positions        wherein the substitution alters the charge at that position to        make the charge more negative or less positive compared to the        precursor protease;    -   c) testing the variant to determine its effectiveness in a high,        medium and low detergent concentration system compared to the        precursor protease; and    -   d) repeating steps a)–c) as necessary to produce a protease        variant that is more effective in a low, medium and high        detergent concentration system than a precursor protease wherein        steps a) and b) can be done in any order.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B-1 through 1B-3 depict the DNA (SEQ ID NO. 1) and aminoacid sequence (SEQ ID NO. 2) for Bacillus amyloliquefaciens subtilisinand a partial restriction map of this gene.

FIG. 2 depicts the conserved amino acid residues among subtilisins fromBacillus amyloliquefaciens (BPN)′ and Bacillus lentus (wild-type).

FIGS. 3A and 3B depict the amino acid sequence of four subtilisins. Thetop line represents the amino acid sequence of subtilisin from Bacillusamyloliquefaciens subtilisin (Also sometimes referred to as subtilisinBPN′) (SEQ ID NO. 3). The second line depicts the amino acid sequence ofsubtilisin from Bacillus subtilis (SEQ ID NO. 4). The third line depictsthe amino acid sequence of subtilisin from B. lichenformis (SEQ D NO.5). The fourth line depicts the amino acid sequence of subtilisin fromBacillus lentus (Also referred to as subtilisin 309 in PCT WO89/06276)(SEQ ID NO. 6). The symbol * denotes the absence of specific amino acidresidues as compared to subtilisin BPN′.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, certain geographies have certain wash conditions and, assuch, use different types of detergents. For example, Japan uses a lowdetergent concentration system while Europe uses a high detergentconcentration system. As discussed previously, the United States uses amedium detergent concentration system. We have found that differentprotease variants perform optimally in these different detergentformulations. However, as a result of these observations, one wouldexpect that it would be impossible to find a protease that would workwell in all three types of detergents. Surprisingly, this is not thecase. A method of rationally designing a protease variant to be used ineither a low detergent concentration system or a high detergentconcentration system or even a medium detergent concentration system aswell as one that works in all three detergent concentration systems hasbeen developed.

We have found that in order to produce a protease variant that is moreefficacious in a low detergent concentration system, it is necessary toreplace positively charged residue(s) either with negatively chargedresidue(s) or neutral residue(s) and/or neutral residue(s) withnegatively charged residue(s). In contrast, we note that in order toproduce a protease variant that is more efficacious in a high detergentconcentration system, it is necessary to replace negatively chargedresidue(s) either with positively charged residue(s) or neutralresidue(s) and/or neutral residue(s) with positively charged residue(s).Further, we have found that many of the protease variants useful in thelow detergent concentration system and/or the high detergentconcentration system also are effective in a medium detergentconcentration system. By balancing these changes, it is possible toproduce a protease variant that works well in low detergentconcentration systems, low and medium detergent concentration systems,medium and high detergent concentration systems, high detergentconcentration systems, or all three detergent concentration systems.

The electrostatic charge of any ionizable amino acid side chain with anacidic or basic function assumes in aqueous solution is a function ofthe pH. The acidic residues Glu and Asp, in an equilibrium process, losea proton by dissociation between pH 3 and 6 thereby acquiring a negativecharge. In a similar fashion, His, Lys, and Arg gradually deprotonatebetween pH 5 and 8, pH 8.5 and 11.5, and pH 11 and 14, respectively,thereby losing a positive charge. The proton of Tyr OH increasinglydissociates between pH 8.5 and 11.5, whereby Tyr acquires a negativecharge. The dissociation range for the carboxy terminus is pH 1 to 4,yielding a negative charge, and for the amino terminus it is pH 8 to 11,accompanied by the loss of a positive charge. The dissociation range foramino acid side chains given here are average values for many proteinsbut they are known to be affected by unusual structural configurationsin some proteins.

The cumulative effect of all charges determines whether a protein has anet positive or net negative charge at a given pH. The pH at whichpositive and negative charges are equally effective and convey anelectrostatically neutral state to a protein is called the isoelectricpoint (pI). A protein will lose or gain charge when the pH is shifted orwhen an amino acid with an ionizable side chain residue is added orremoved. An increase in net positive charge can be achieved either byreplacing a residue that at a given pH is negatively charged with anuncharged or a positively charged residue, leading to a formal chargechange of +1 and +2, respectively. By replacing an uncharged side chainresidue with one that is protonated at the given pH the formal chargechange would be +1. Similarly, net negative charge can be increased byreplacing positively and uncharged side chains with negatively chargedside chains at the pH of observation and gain a formal in crease ofnegative charge by −1 and −2, respectively.

A low detergent concentration system includes detergents where less thanabout 800 ppm of detergent components are present in the wash water.Japanese detergents are typically considered low detergent concentrationsystem as they have approximately 667 ppm of detergent componentspresent in the wash water.

A medium detergent concentration includes detergents where between about800 ppm and about 2000 ppm of detergent components are present in thewash water. North American detergents are generally considered to bemedium detergent concentration systems as they have approximately 975ppm of detergent components present in the wash water. Brazil typicallyhas approximately 1500 ppm of detergent components present in the washwater.

A high detergent concentration system includes detergents where greaterthan about 2000 ppm of detergent components are present in the washwater. European detergents are generally considered to be high detergentconcentration systems as they have approximately 4500–5000 ppm ofdetergent components in the wash water.

Latin American detergents are generally high suds phosphate builderdetergents and the range of detergents used in Latin America can fall inboth the medium and high detergent concentrations as they range from1500 ppm to 6000 ppm of detergent components in the wash water. Asmentioned above, Brazil typically has approximately 1500 ppm ofdetergent components present in the wash water. However, other high sudsphosphate builder detergent geographies, not limited to other LatinAmerican countries, may have high detergent concentration systems up toabout 6000 ppm of detergent components present in the wash water.

In light of the foregoing, it is evident that concentrations ofdetergent compositions in typical wash solutions throughout the worldvaries from less than about 800 ppm of detergent composition (“lowdetergent concentration geographies”), for example about 667 ppm inJapan, to between about 800 ppm to about 2000 ppm (“medium detergentconcentration geographies”), for example about 975 ppm in U.S. and about1500 ppm in Brazil, to greater than about 2000 ppm (“high detergentconcentration geographies”), for example about 4500 ppm to about 5000ppm in Europe and about 6000 ppm in high suds phosphate buildergeographies.

The concentrations of the typical wash solutions are determinedempirically. For example, in the U.S., a typical washing machine holds avolume of about 64.4 L of wash solution. Accordingly, in order to obtaina concentration of about 975 ppm of detergent within the wash solutionabout 62.79 g of detergent composition must be added to the 64.4 L ofwash solution. This amount is the typical amount measured into the washwater by the consumer using the measuring cup provided with thedetergent.

Proteases generally act to cleave peptide bands of proteins or peptides.As used herein, “protease” means a naturally-occurring protease or arecombinant protease. Naturally-occurring proteases includeα-aminoacylpeptide hydrolase, peptidylamino acid hydrolase, acylaminohydrolase, serine carboxypeptidase, metallocarboxypeptidase, thiolproteinase, carboxylproteinase and metalloproteinase. Serine, metallo,thiol and acid proteases are included, as well as endo andexo-proteases.

The present invention includes protease enzymes which are non-naturallyoccurring variants (protease variants) having a different proteolyticactivity, stability, substrate specificity, pH profile and/orperformance characteristic as compared to the precursor protease fromwhich the amino acid sequence of the variant is derived. Specifically,such protease variants have an amino and sequence not found in nature,which is derived by substitution of a plurality of amino acid residuesof a precursor protease with different amino acids. The precursorprotease may be a naturally-occurring protease or recombinant protease.

The protease variants useful herein encompass the substitution of any ofthe nineteen naturally occurring L-amino acids at the designated aminoacid residue positions. Such substitutions can be made in any precursorsubtilisin (procaryotic, eucaryotic, mammalian, etc.). Throughout thisapplication reference is made to various amino acids by way of commonone- and three-letter codes. Such codes are identified in Dale, M. W.(1989), Molecular Genetics of Bacteria, John Wiley & Sons, Ltd.,Appendix B.

The protease variants useful herein are preferably derived from aBacillus subtilisin. More preferably, the protease variants are derivedfrom Bacillus lentus subtilisin and/or subtilisin 309.

Subtilisins are bacterial or fungal proteases which generally act tocleave peptide bonds of proteins or peptides. As used herein,“subtilisin” means a naturally-occurring subtilisin or a recombinantsubtilisin. A series of naturally-occurring subtilisins is known to beproduced and often secreted by various microbial species. Amino acidsequences of the members of this series are not entirely homologous.However, the subtilisins in this series exhibit the same or similar typeof proteolytic activity. This class of serine proteases shares a commonamino acid sequence defining a catalytic triad which distinguishes themfrom the chymotrypsin related class of serine proteases. The subtilisinsand chymotrypsin related serine proteases both have a catalytic triadcomprising aspartate, histidine and serine. In the subtilisin relatedproteases the relative order of these amino acids, reading from theamino to carboxy terminus, is aspartate-histidine-serine. In thechymotrypsin related proteases, the relative order, however, ishistidine-aspartate-serine. Thus, subtilisin herein refers to a serineprotease having the catalytic triad of subtilisin related proteases.Examples include but are not limited to the subtilisins identified inFIG. 3 herein. Generally and for purposes of the present invention,numbering of the amino acids in proteases corresponds to the numbersassigned to the mature Bacillus amyloliquefaciens subtilisin sequencepresented in FIG. 1.

“Recombinant subtilisin” or “recombinant protease” refer to a subtilisinor protease in which the DNA sequence encoding the subtilisin orprotease is modified to produce a variant (or mutant) DNA sequence whichencodes the substitution, deletion or insertion of one or more aminoacids in the naturally-occurring amino acid sequence. Suitable methodsto produce such modification, and which may be combined with thosedisclosed herein, include those disclosed in U.S. Pat. No. RE 34,606,U.S. Pat. Nos. 5,204,015 and 5,185,258, 5,700,676, 5,801,038, and5,763,257.

“Non-human subtilisins” and the DNA encoding them may be obtained frommany procaryotic and eucaryotic organisms. Suitable examples ofprocaryotic organisms include gram negative organisms such as E. coli orPseudomonas and gram positive bacteria such as Micrococcus or Bacillus.Examples of eucaryotic organisms from which subtilisin and their genesmay be obtained include yeast such as Saccharomyces cerevisiae, fungisuch as Aspergillus sp.

A “protease variant” has an amino acid sequence which is derived fromthe amino acid sequence of a “precursor protease”. The precursorproteases include naturally-occurring proteases and recombinantproteases. The amino acid sequence of the protease variant is “derived”from the precursor protease amino acid sequence by the substitution,deletion or insertion of one or more amino acids of the precursor aminoacid sequence. Such modification is of the “precursor DNA sequence”which encodes the amino acid sequence of the precursor protease ratherthan manipulation of the precursor protease enzyme per se. Suitablemethods for such manipulation of the precursor DNA sequence includemethods disclosed herein, as well as methods known to those skilled inthe art (see, for example, EP 0 328299, WO89/06279 and the U.S. patentsand applications already referenced herein).

These amino acid position numbers refer to those assigned to the matureBacillus amyloliquefaciens subtilisin sequence presented in FIG. 1. Theinvention, however, is not limited to the mutation of this particularsubtilisin but extends to precursor proteases containing amino acidresidues at positions which are “equivalent” to the particularidentified residues in Bacillus amyloliquefaciens subtilisin. In apreferred embodiment of the present invention, the precursor protease isBacillus lentus subtilisin and the substitutions are made at theequivalent amino acid residue positions in B. Lentus corresponding tothose listed above.

A residue (amino acid) position of a precursor protease is equivalent toa residue of Bacillus amyloliquefaciens subtilisin if it is eitherhomologous (i.e., corresponding in position in either primary ortertiary structure) or analogous to a specific residue or portion ofthat residue in Bacillus amyloliquefaciens subtilisin (i.e., having thesame or similar functional capacity to combine, react, or interactchemically).

In order to establish homology to primary structure, the amino acidsequence of a precursor protease is directly compared to the Bacillusamyloliquefaciens subtilisin primary sequence and particularly to a setof residues known to be invariant in subtilisins for which sequence isknown. For example, FIG. 2 herein shows the conserved residues asbetween B. amyloliquefaciens subtilisin and B. lentus subtilisin. Afteraligning the conserved residues, allowing for necessary insertions anddeletions in order to maintain alignment (i.e., avoiding the eliminationof conserved residues through arbitrary deletion and insertion), theresidues equivalent to particular amino acids in the primary sequence ofBacillus amyloliquefaciens subtilisin are defined. Alignment ofconserved residues preferably should conserve 100% of such residues.However, alignment of greater than 75% or as little as 50% of conservedresidues is also adequate to define equivalent residues. Conservation ofthe catalytic triad, Asp32/His64/Ser221 should be maintained. Siezen etal. (1991) Protein Eng. 4(7):719–737 shows the alignment of a largenumber of serine proteases. Siezen et al. refer to the grouping assubtilases or subtilisin-like serine proteases.

For example, in FIG. 3, the amino acid sequence of subtilisin fromBacillus amyloliquefaciens, Bacillus subtilis, Bacillus licheniformis(carlsbergensis) and Bacillus lentus are aligned to provide the maximumamount of homology between amino acid sequences. A comparison of thesesequences shows that there are a number of conserved residues containedin each sequence. These conserved residues (as between BPN′ and B.lentus) are identified in FIG. 2.

These conserved residues, thus, may be used to define the correspondingequivalent amino acid residues of Bacillus amyloliquefaciens subtilisinin other subtilisins such as subtilisin from Bacillus lentus (PCTPublication No. WO89/06279 published Jul. 13, 1989), the preferredprotease precursor enzyme herein, or the subtilisin referred to as PB92(EP 0 328 299), which is highly homologous to the preferred Bacilluslentus subtilisin. The amino acid sequences of certain of thesesubtilisins are aligned in FIGS. 3A and 3B with the sequence of Bacillusamyloliquefaciens subtilisin to produce the maximum homology ofconserved residues. As can be seen, there are a number of deletions inthe sequence of Bacillus lentus as compared to Bacillusamyloliquefaciens subtilisin. Thus, for example, the equivalent aminoacid for Val165 in Bacillus amyloliquefaciens subtilisin in the othersubtilisins is isoleucine for B. lentus and B. licheniformis.

“Equivalent residues” may also be defined by determining homology at thelevel of tertiary structure for a precursor protease whose tertiarystructure has been determined by x-ray crystallography. Equivalentresidues are defined as those for which the atomic coordinates of two ormore of the main chain atoms of a particular amino acid residue of theprecursor protease and Bacillus amyloliquefaciens subtilisin (N on N, CAon CA, C on C and O on O) are within 0.13 nm and preferably 0.1 nm afteralignment. Alignment is achieved after the best model has been orientedand positioned to give the maximum overlap of atomic coordinates ofnon-hydrogen protein atoms of the protease in question to the Bacillusamyloliquefaciens subtilisin. The best model is the crystallographicmodel giving the lowest R factor for experimental diffraction data atthe highest resolution available.

${R\mspace{11mu}{factor}} = \frac{{\Sigma_{h}{{{Fo}(h)}}} - {{{Fc}(h)}}}{\Sigma_{h}{{{Fo}(h)}}}$

Equivalent residues which are functionally analogous to a specificresidue of Bacillus amyloliquefaciens subtilisin are defined as thoseamino acids of the precursor protease which may adopt a conformationsuch that they either alter, modify or contribute to protein structure,substrate binding or catalysis in a manner defined and attributed to aspecific residue of the Bacillus amyloliquefaciens subtilisin. Further,they are those residues of the precursor protease (for which a tertiarystructure has been obtained by x-ray crystallography) which occupy ananalogous position to the extent that, although the main chain atoms ofthe given residue may not satisfy the criteria of equivalence on thebasis of occupying a homologous position, the atomic coordinates of atleast two of the side chain atoms of the residue lie with 0.13 nm of thecorresponding side chain atoms of Bacillus amyloliquefaciens subtilisin.The coordinates of the three dimensional structure of Bacillusamyloliquefaciens subtilisin are set forth in EPO Publication No. 0 251446 (equivalent to U.S. Pat. No. 5,182,204, the disclosure of which isincorporated herein by reference) and can be used as outlined above todetermine equivalent residues on the level of tertiary structure.

Some of the residues identified for substitution are conserved residueswhereas others are not. In the case of residues which are not conserved,the substitution of one or more amino acids is limited to substitutionswhich produce a variant which has an amino acid sequence that does notcorrespond to one found in nature. In the case of conserved residues,such substitutions should not result in a naturally-occurring sequence.The protease variants of the present invention include the mature formsof protease variants, as well as the pro- and prepro-forms of suchprotease variants. The prepro-forms are the preferred construction sincethis facilitates the expression, secretion and maturation of theprotease variants.

“Prosequence” refers to a sequence of amino acids bound to theN-terminal portion of the mature form of a protease which when removedresults in the appearance of the “mature” form of the protease. Manyproteolytic enzymes are found in nature as translational proenzymeproducts and, in the absence of post-translational processing, areexpressed in this fashion. A preferred prosequence for producingprotease variants is the putative prosequence of Bacillusamyloliquefaciens subtilisin, although other protease prosequences maybe used.

A “signal sequence” or “presequence” refers to any sequence of aminoacids bound to the N-terminal portion of a protease or to the N-terminalportion of a proprotease which may participate in the secretion of themature or pro forms of the protease. This definition of signal sequenceis a functional one, meant to include all those amino acid sequencesencoded by the N-terminal portion of the protease gene which participatein the effectuation of the secretion of protease under nativeconditions. The present invention utilizes such sequences to effect thesecretion of the protease variants as defined herein. One possiblesignal sequence comprises the first seven amino acid residues of thesignal sequence from Bacillus subtilis subtilisin fused to the remainderof the signal sequence of the subtilisin from Bacillus lentus (ATCC21536).

A “prepro” form of a protease variant consists of the mature form of theprotease having a prosequence operably linked to the amino terminus ofthe protease and a “pre” or “signal” sequence operably linked to theamino terminus of the prosequence.

“Expression vector” refers to a DNA construct containing a DNA sequencewhich is operably linked to a suitable control sequence capable ofeffecting the expression of said DNA in a suitable host. Such controlsequences include a promoter to effect transcription, an optionaloperator sequence to control such transcription, a sequence encodingsuitable mRNA ribosome binding sites and sequences which controltermination of transcription and translation. The vector may be aplasmid, a phage particle, or simply a potential genomic insert. Oncetransformed into a suitable host, the vector may replicate and functionindependently of the host genome, or may, in some instances, integrateinto the genome itself. In the present specification, “plasmid” and“vector” are sometimes used interchangeably as the plasmid is the mostcommonly used form of vector at present. However, the invention isintended to include such other forms of expression vectors which serveequivalent functions and which are, or become, known in the art.

The “host cells” used in the present invention generally are procaryoticor eucaryotic hosts which preferably have been manipulated by themethods disclosed in U.S. Pat. No. RE 34,606 to render them incapable ofsecreting enzymatically active endoprotease. A preferred host cell forexpressing protease is the Bacillus strain BG2036 which is deficient inenzymatically active neutral protease and alkaline protease(subtilisin). The construction of strain BG2036 is described in detailin U.S. Pat. No. 5,264,366. Other host cells for expressing proteaseinclude Bacillus subtilis I168 (also described in U.S. Pat. No. RE34,606 and U.S. Pat. No. 5,264,366, the disclosure of which areincorporated herein by reference), as well as any suitable Bacillusstrain such as B. licheniformis, B. lentus, etc.

Host cells are transformed or transfected with vectors constructed usingrecombinant DNA techniques. Such transformed host cells are capable ofeither replicating vectors encoding the protease variants or expressingthe desired protease variant. In the case of vectors which encode thepre- or prepro-form of the protease variant, such variants, whenexpressed, are typically secreted from the host cell into the host cellmedium.

“Operably linked,” when describing the relationship between two DNAregions, simply means that they are functionally related to each other.For example, a presequence is operably linked to a peptide if itfunctions as a signal sequence, participating in the secretion of themature form of the protein most probably involving cleavage of thesignal sequence. A promoter is operably linked to a coding sequence ifit controls the transcription of the sequence; a ribosome binding siteis operably linked to a coding sequence if it is positioned so as topermit translation.

The genes encoding the naturally-occurring precursor protease may beobtained in accord with the general methods known to those skilled inthe art. The methods generally comprise synthesizing labeled probeshaving putative sequences encoding regions of the protease of interest,preparing genomic libraries from organisms expressing the protease, andscreening the libraries for the gene of interest by hybridization to theprobes. Positively hybridizing clones are then mapped and sequenced.

The cloned protease is then used to transform a host cell in order toexpress the protease. The protease gene is then ligated into a high copynumber plasmid. This plasmid replicates in hosts in the sense that itcontains the well-known elements necessary for plasmid replication: apromoter operably linked to the gene in question (which may be suppliedas the gene's own homologous promoter if it is recognized, i.e.,transcribed, by the host), a transcription termination andpolyadenylation region (necessary for stability of the mRNA transcribedby the host from the protease gene in certain eucaryotic host cells)which is exogenous or is supplied by the endogenous terminator region ofthe protease gene and, desirably, a selection gene such as an antibioticresistance gene that enables continuous cultural maintenance ofplasmid-infected host cells by growth in antibiotic-containing media.High copy number plasmids also contain an origin of replication for thehost, thereby enabling large numbers of plasmids to be generated in thecytoplasm without chromosomal limitations. However, it is within thescope herein to integrate multiple copies of the protease gene into hostgenome. This is facilitated by procaryotic and eucaryotic organismswhich are particularly susceptible to homologous recombination.

The gene can be a natural B. lentus gene. Alternatively, a syntheticgene encoding a naturally-occurring or mutant precursor protease may beproduced. In such an approach, the DNA and/or amino acid sequence of theprecursor protease is determined. Multiple, overlapping syntheticsingle-stranded DNA fragments are thereafter synthesized, which uponhybridization and ligation produce a synthetic DNA encoding theprecursor protease. An example of synthetic gene construction is setforth in Example 3 of U.S. Pat. No. 5,204,015, the disclosure of whichis incorporated herein by reference.

Once the naturally-occurring or synthetic precursor protease gene hasbeen cloned, a number of modifications are undertaken to enhance the useof the gene beyond synthesis of the naturally-occurring precursorprotease. Such modifications include the production of recombinantproteases as disclosed in U.S. Pat. No. RE 34,606 and EPO PublicationNo. 0 251 446 and the production of protease variants described herein.

The following cassette mutagenesis method may be used to facilitate theconstruction of the protease variants of the present invention, althoughother methods may be used. First, the naturally-occurring gene encodingthe protease is obtained and sequenced in whole or in part. Then thesequence is scanned for a point at which it is desired to make amutation (deletion, insertion or substitution) of one or more aminoacids in the encoded enzyme. The sequences flanking this point areevaluated for the presence of restriction sites for replacing a shortsegment of the gene with an oligonucleotide pool which when expressedwill encode various mutants. Such restriction sites are preferablyunique sites within the protease gene so as to facilitate thereplacement of the gene segment. However, any convenient restrictionsite which is not overly redundant in the protease gene may be used,provided the gene fragments generated by restriction digestion can bereassembled in proper sequence. If restriction sites are not present atlocations within a convenient distance from the selected point (from 10to 15 nucleotides), such sites are generated by substituting nucleotidesin the gene in such a fashion that neither the reading frame nor theamino acids encoded are changed in the final construction. Mutation ofthe gene in order to change its sequence to conform to the desiredsequence is accomplished by M13 primer extension in accord withgenerally known methods. The task of locating suitable flanking regionsand evaluating the needed changes to arrive at two convenientrestriction site sequences is made routine by the redundancy of thegenetic code, a restriction enzyme map of the gene and the large numberof different restriction enzymes. Note that if a convenient flankingrestriction site is available, the above method need be used only inconnection with the flanking region which does not contain a site.

Once the naturally-occurring DNA or synthetic DNA is cloned, therestriction sites flanking the positions to be mutated are digested withthe cognate restriction enzymes and a plurality of endtermini-complementary oligonucleotide cassettes are ligated into thegene. The mutagenesis is simplified by this method because all of theoligonucleotides can be synthesized so as to have the same restrictionsites, and no synthetic linkers are necessary to create the restrictionsites.

As used herein, proteolytic activity is defined as the rate ofhydrolysis of peptide bonds per milligram of active enzyme. Many wellknown procedures exist for measuring proteolytic activity (K. M. Kalisz,“Microbial Proteinases,” Advances in BiochemicalEngineering/Biotechnology, A. Fiechter ed., 1988). In addition to or asan alternative to modified proteolytic activity, the variant enzymes ofthe present invention may have other modified properties such as K_(m),k_(cat), k_(cat)/K_(m) ratio and/or modified substrate specificityand/or modified pH activity profile. These enzymes can be tailored forthe particular substrate which is anticipated to be present, forexample, in the preparation of peptides or for hydrolytic processes suchas laundry uses.

In one aspect of the invention, the objective is to secure a variantprotease having altered proteolytic activity as compared to theprecursor protease, since increasing such activity (numerically larger)enables the use of the enzyme to more efficiently act on a targetsubstrate. Also of interest are variant enzymes having altered thermalstability and/or altered substrate specificity as compared to theprecursor. In some instances, lower proteolytic activity may bedesirable, for example a decrease in proteolytic activity would beuseful where the synthetic activity of the proteases is desired (as forsynthesizing peptides). One may wish to decrease this proteolyticactivity, which is capable of destroying the product of such synthesis.Conversely, in some instances it may be desirable to increase theproteolytic activity of the variant enzyme versus its precursor.Additionally, increases or decreases (alteration) of the stability ofthe variant, whether alkaline or thermal stability, may be desirable.Increases or decreases in k_(cat), K_(m) or k_(cat)/K_(m) are specificto the substrate used to determine these kinetic parameters.

In another aspect of the invention, it has been found that proteasevariants containing substitutions of the amino acids at one or moreresidue positions so that the substitution alters the charge at thatposition to make the charge more negative or less positive compared to aprecursor protease are more effective in a low detergent concentrationthan a precursor protease.

In a further aspect of the invention, it has been found that proteasevariants containing substitutions of the amino acids at one or moreresidue positions so that the substitution alters the charge at thatposition to make the charge more positive or less negative compared to aprecursor protease are more effective in a high detergent concentrationthan a precursor protease.

Further, we have found that many of the protease variants useful in thelow detergent concentration system and/or the high detergentconcentration system also are effective in a medium detergentconcentration system.

These substitutions are preferably made in Bacillus lentus (recombinantor native-type) subtilisin, although the substitutions may be made inany Bacillus protease, preferably Bacillus subtilisins.

Based on the screening results obtained with the variant proteases, thenoted mutations in Bacillus amyloliquefaciens subtilisin are importantto the proteolytic activity, performance and/or stability of theseenzymes and the cleaning or wash performance of such variant enzymes.

Many of the protease variants of the invention are useful in formulatingvarious detergent compositions or personal care formulations such asshampoos or lotions. A number of known compounds are suitablesurfactants useful in compositions comprising the protease mutants ofthe invention. These include nonionic, anionic, cationic or zwitterionicdetergents, as disclosed in U.S. Pat. No. 4,404,128 to Barry J. Andersonand U.S. Pat No. 4,261,868 to Jiri Flora, et al. A suitable detergentformulation is that described in Example 7 of U.S. Pat. No. 5,204,015(previously incorporated by reference). The art is familiar with thedifferent formulations which can be used as cleaning compositions. Inaddition to typical cleaning compositions, it is readily understood thatthe protease variants of the present invention may be used for anypurpose that native or wild-type proteases are used. Thus, thesevariants can be used, for example, in bar or liquid soap applications,dishcare formulations, contact lens cleaning solutions or products,peptide hydrolysis, waste treatment, textile applications, asfusion-cleavage enzymes in protein production, etc. The variants of thepresent invention may comprise enhanced performance in a detergentcomposition (as compared to the precursor). As used herein, enhancedperformance in a detergent is defined as increasing cleaning of certainenzyme sensitive stains such as grass or blood, as determined by usualevaluation after a standard wash cycle.

Proteases of the invention can be formulated into known powdered andliquid detergents having pH between 6.5 and 12.0 at levels of about 0.01to about 5% (preferably 0.1% to 0.5%) by weight. These detergentcleaning compositions can also include other enzymes such as knownproteases, amylases, cellulases, lipases or endoglycosidases, as well asbuilders and stabilizers.

The addition of proteases of the invention to conventional cleaningcompositions does not create any special use limitation. In other words,any temperature and pH suitable for the detergent is also suitable forthe present compositions as long as the pH is within the above range,and the temperature is below the described protease's denaturingtemperature. In addition, proteases of the invention can be used in acleaning composition without detergents, again either alone or incombination with builders and stabilizers.

The present invention also relates to cleaning compositions containingthe protease variants of the invention. The cleaning compositions mayadditionally contain additives which are commonly used in cleaningcompositions. These can be selected from, but not limited to, bleaches,surfactants, builders, enzymes and bleach catalysts. It would be readilyapparent to one of ordinary skill in the art what additives are suitablefor inclusion into the compositions. The list provided herein is by nomeans exhaustive and should be only taken as examples of suitableadditives. It will also be readily apparent to one of ordinary skill inthe art to only use those additives which are compatible with theenzymes and other components in the composition, for example,surfactant.

When present, the amount of additive present in the cleaning compositionis from about 0.01% to about 99.9%, preferably about 1% to about 95%,more preferably about 1% to about 80%.

The variant proteases of the present invention can be included in animalfeed such as part of animal feed additives as described in, for example,U.S. Pat. Nos. 5,612,055; 5,314,692; and 5,147,642.

One aspect of the invention is a composition for the treatment of atextile that includes variant proteases of the present invention. Thecomposition can be used to treat for example silk or wool as describedin publications such as RD 216,034; EP 134,267; U.S. Pat. No. 4,533,359;and EP 344,259.

The following is presented by way of example and is not to be construedas a limitation to the scope of the claims.

All publications and patents referenced herein are hereby incorporatedby reference in their entirety.

EXAMPLE 1

A large number of protease variants were produced and purified usingmethods well known in the art. All mutations were made in Bacilluslentus GG36 subtilisin.

The protease variants produced were tested for performance in two typesof detergent and wash conditions using a microswatch assay described in“An improved method of assaying for a preferred enzyme and/or preferreddetergent composition”, U.S. Ser. No. 09/554,992 which claims priorityto U.S. Ser. No. 60/068,796 filed Dec. 24, 1997 and published asInternational Application No. WO 99/34011.

Tables 1–13 list the variant proteases assayed and the results oftesting in two different detergents. All values are given as comparisonto the first protease shown in the table (i.e., a value of 1.32indicates an ability to release 132% of the stain as opposed to the 100%of the first variant in the table).

Column A shows the charge difference of a variant. For column B, thedetergent was 0.67 g/l filtered Ariel Ultra (Procter & Gamble,Cincinnati, Ohio, USA), in a solution containing 3 grains per gallonmixed Ca²⁺/Mg²⁺ hardness, and 0.3 ppm enzyme was used in each well at25° C. (low concentration detergent system). For column C, the detergentwas 3.38 g/l filtered Ariel Futur (Procter & Gamble, Cincinnati, Ohio,USA), in a solution containing 15 grains per gallon mixed Ca²⁺/Mg²⁺hardness, and 0.3 ppm enzyme was used in each well at 40° C. (highconcentration detergent system).

TABLE 1 A B C N76D S103A V1041 Q109R 1.00 1.00 N76D S103A V1041 Q109RQ245R +1 0.48 1.41

TABLE 2 A B C V68A N76D S103A V1041 G159D Q236H Q245R 1.00 1.00 V68AN76D S103A V1041 G159D N204D Q236H Q245R −1 1.11 0.03

TABLE 3 A B C V68A N76D S103A V104I 1.00 1.00 T22K V68A N76D S103A V104I+1 0.74 1.85

TABLE 4 A B C N76D S103A V104I M222S 1.00 1.00 N76D S103A V104I N173RM222S 0 0.66 1.84 Q12R N76D S103A V104I M222S Q245R +1 0.41 5.84

TABLE 5 A B C Q12R N76D S103A I104T S130T M222S Q245R 1.00 1.00 Q12RN76D S103A I104T SI30T M222S Q245R N261D −1 1.79 0.81 Q12R N76D S103AI104T S130T R170S N185D M222S N243D Q245R −3 2.87 0.02

TABLE 6 A B C V68A N76D S103A V1041 G159D Q236H 1.00 1.00 V68A N76DS103A V1041 G159D Q236H Q245R +1 0.94 6.80 V68A S103A V104I G159D A232VQ236H Q245R N252K +3 0.44 20.60

TABLE 7 A B C V68A N76D S103A V104I G159D A232V Q236H Q245R 1.00 1.00V68A N76D S103A V104I G159D P210R A232V Q236H Q245R +1 0.44 2.66

TABLE 8 A B C V68A S103A V104I G159D A232V Q236H Q245R N252K 1.00 1.00V68A S103A V104I G159D A232V Q236H Q245R N248D N252K −1 1.96 0.65

TABLE 9 A B C V68A S103A V104I G159D A232V Q236H Q245R 1.00 1.00 V68AS103A V104I G159D A232V Q236H K237E Q245R −2 1.27 0.12

TABLE 10 A B C V68A S103A V104I G159D A232V Q236H Q245R L257V 1.00 1.00V68A N76D S103A V104I G159D A232V Q236H Q245R L257V −1 1.56 0.48

TABLE 11 A B C S103A V104I G159D A232V Q236H Q245R N248D N252K 1.00 1.00S103A V104I G159D L217E A232V Q236H Q245R N248D N252K −1 1.90 0.15

TABLE 12 A B C S103A V104I S101G G159D A232V Q236H Q245R N248D N252K1.00 1.00 N76D S103A V104I S101G G159D A232V Q236H Q245R N248D N252K −11.28 0.39

TABLE 13 A B C N62D S103A V104I G159D T213R A232V Q236H Q245R N248DN252K 1.00 1.00 N62D S103A V104I Q109R G159D T213R A232V Q236H Q245RN248D N252K +1 0.40 1.74

EXAMPLE 2

The following protease variants were made and tested as noted in Example1.

The variants in Table 14 are protease variants which have both types ofsubstitutions: those which alter the charge at a position to make thecharge more negative or less positive and those which alter the chargeat a position to make the charge more positive or less negative comparedto B. lentus GG36 as well as neutral substitutions that do not affectthe charge at a given residue position. This produces protease variantsthat perform better than a standard in both low detergent concentrationsystems (column A; 0.67 g/l filtered Ariel Ultra (Procter & Gamble,Cincinnati, Ohio, USA), in a solution containing 3 grains per gallonmixed Ca²⁺/Mg²⁺ hardness, and 0.3 ppm enzyme was used in each well at25° C.) and high detergent concentration systems (column B; 3.38 g/lfiltered Ariel Futur (Procter & Gamble, Cincinnati, Ohio, USA), in asolution containing 15 grains per gallon mixed Ca²⁺/Mg²⁺ hardness, and0.3 ppm enzyme was used in each well at 40° C.).

TABLE 14 A B N76D S103A V104I 1.00 1.00 V68A S103A V104I G159D A232VQ236H Q245R N252K 1.41 1.85 V68A N76D S103A V104I G159D T213R A232VQ236H Q245R T260A 1.30 1.73 V68A S103A V104I G159D A232V Q236H Q245RN248D N252K 2.77 1.20 V68A S103A V104I N140D G159D A232V Q236H Q245RN252K 2.96 1.42 N43K V68A S103A V104I G159D A232V Q236H Q245R 2.05 1.78N43D V68A S103A V104I G159D A232V Q236H Q245R N252K 2.00 1.34 V68A N76DS103A V104I G159D A215R A232V Q236H Q245R 1.67 1.45 Q12R V68A N76D S103AV104I G159D A232V Q236H Q245R 2.16 1.72 N76D S103A V104I V147I G159DA232V Q236H Q245R N248S K251R 1.35 1.29 V68A N76D S103A V104I G159DA232V Q236H Q245R S256R 2.01 1.72 V68A N76D S103A V104I G159D Q206RA232V Q236H Q245R 2.09 1.62 S103A V104I G159D A232V Q236H Q245R N248DN252K 1.44 1.41 G20R V68A S103A V104I G159D A232V Q236H Q245R N248DN252K 1.81 1.72 V68A S103A V104I G159D A232V Q236H Q245R N248D N252KL257R 1.51 1.41 V68A S103A V104I A232V Q236H Q245R N248D N252K 1.04 1.50N76D S103A V104I G159D A232V Q236H Q245R L257V 1.92 1.09

1. A Bacillus subtilisin variant comprising a substitution of an aminoacid at one or more residue positions of a precursor Bacillus subtilisinwherein said substitution alters the overall charge of the precursorsubtilisin resulting in a subtilisin variant that is more positive orless negative relative to the precursor subtilisin, wherein saidsubstitution makes the subtilisin variant more effective than theprecursor in a detergent concentration system wherein the detergentcomponents are primarily negatively charged or where the overall chargebalance of the detergent components is not positive, and wherein saidsubstitutions are selected from the group consisting ofN76D/S103A/V104I/Q109R/Q245R, Q12R/N76D/S103A/V104I/M222S/Q245R,V68A/S103A/V104I/G159D/A232V/Q236H/Q245R/N252K,V68A/N76D/S103A/V104I/G159D/P210R/A232V/Q236H/Q245R, andN62D/S103A/V104I/Q109R/G159D/T213R/M232V/Q236H/Q245R/N248D/N252K, andwherein said positions are numbered according to the positions of themature subtilisin BPN′ having the amino acid sequence set forth in SEQID NO:3.
 2. A Bacillus subtilisin variant comprising a substitution ofan amino acid at one or more residue positions of a precursor Bacillussubtilisin wherein said variants comprise the sets of substitutionsT22K/V68A/N76D/S103A/V104I or V68A/N76D/S103A/V104I/G159D/Q236H/Q245R,wherein said positions are numbered according to the positions of themature subtilisin BPN′ having the amino acid sequence set forth in SEQID NO:3.
 3. The subtilisin variant according to claim 1 wherein saidprecursor subtilisin is a Bacillus lentus subtilisin.
 4. A cleaningcomposition comprising the subtilisin variant of claim 1.